RT, qPCR
RT, qPCR kdorfman Tue, 04/26/2022 - 14:16Separate RT & qPCR
RT 10 groups @ 6 rxns 2 groups @ 8 rxns
Equipment
Hot blocks at
- 50°C (2 dry baths - this is the long step and there are at least 4x as many tubes as groups),
- 65°C
- 70°C
reagent uL/rxn uL/8 rxn (+ xtra) uL/class notes Oligo dT 50 µM 1 1 15 180 10 mM dNTP mix 2 1 15 180 RNAse-free water 11 + 40 60 720 aliquot once for both RT and qPCR 5X 1st strand buffer 4 60 720 comes with Superscript 0.1M DTT 1 15 125 1500 | comes with Superscript RNaseOUT 3 1 8 96 Dispensed from freezer box during lab Superscript 4 1 8 96 Dispensed from freezer box during lab
864 rxns @ 10uL
(24 x 3 rxns per group x 12 groups)
Reagent | uL per rxn | uL per 75 rxns |
---|---|---|
2x SYBR Green master mix5 | 5 uL | 375 uL |
water | <5 uL | 375 uL |
forward primer | 0.5 uL | |
reverse primer | 0.5 uL | |
cDNA | ~1 uL |
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Fisher FERSO131 Oligo dT 100 µM (=0.5µg/µL) 60 µL @ ~$50. Dilute to 50µM!! (for emergencies: 50 µM oligo(dT) Fisher stockroom: C1101 0.5µg/µL x 40µL = 20µg) - Order T(18) from Invitrogen??? ↩︎
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14 µL each + 84 µL RNase-free water OR Fisher FERR0192 ↩︎
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Invitrogen 10777019 RNAse out 5KU @ $143 ↩︎
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superscript III 200U/µL Invitrogen 18080044 10KU @ $259
qPCR ↩︎
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Thermo Fisher 4385612 ↩︎